Jennifer M. Golas

SKI-606, a Src/Abl Inhibitor with In vivo Activity in Colon Tumor Xenograft Models

Src up-regulation is a common event in human cancers. In colorectal cancer, increased Src levels are an indicator of poor prognosis, and progression to metastatic disease is associated with substantial increases in Src activity. Therefore, we examined the activity of SKI-606, a potent inhibitor of Src and Abl kinases, against colon tumor lines in vitro and in s.c. tumor xenograft models. SKI-606 inhibited Src autophosphorylation with an IC(50) of approximately 0.25 micromol/L in HT29 cells. Phosphorylation of Tyr(925) of focal adhesion kinase, a Src substrate, was reduced by similar concentrations of inhibitor. Antiproliferative activity on plastic did not correlate with Src inhibition in either HT29 or Colo205 cells (IC(50)s, 1.5 and 2.5 micromol/L, respectively), although submicromolar concentrations of SKI-606 inhibited HT29 cell colony formation in soft agar. SKI-606 also caused loosely aggregated Colo205 spheroids to condense into compact spheroids. On oral administration to nude mice at the lowest efficacious dose, peak plasma concentrations of approximately 3 micromol/L, an oral bioavailability of 18%, and a t(1/2) of 8.6 hours were observed. SKI-606 was orally active in s.c. colon tumor xenograft models and caused substantial reductions in Src autophosphorylation on Tyr(418) in HT29 and Colo205 tumors. SKI-606 inhibited HT29 tumor growth on once daily administration, whereas twice daily administration was necessary to inhibit Colo205, HCT116, and DLD1 tumor growth. These results support development of SKI-606 as a therapeutic agent for treatment of colorectal cancer.

Discovery of benzisoxazoles as potent inhibitors of chaperone heat shock protein 90.

Heat shock protein 90 (Hsp90) is a molecular chaperone that is responsible for activating many signaling proteins and is a promising target in tumor biology. We have identified small-molecule benzisoxazole derivatives as Hsp90 inhibitors. Crystallographic studies show that these compounds bind in the ATP binding pocket interacting with the Asp93. Structure based optimization led to the identification of potent analogues, such as 13, with good biochemical profiles.

Discovery of a stable macrocyclic o-aminobenzamide Hsp90 inhibitor which significantly decreases tumor volume in a mouse xenograft model.

An extension of our previously reported series of macrocyclic ortho-aminobenzamide Hsp90 inhibitors is reported. Addition of a second methyl group to the tether provided analogs that show increased potency in binding as well as cell-proliferation assays and, more importantly, are stable toward microsomes. We wish to disclose the discovery of a macrocycle which showed impressive biomarker activity 24-h post dosing and which demonstrated prolonged exposure in tumors. When studied in a lung cancer xenograft model, the compound demonstrated significant tumor size reduction.

Design and SAR of macrocyclic Hsp90 inhibitors with increased metabolic stability and potent cell-proliferation activity.

A novel series of macrocyclic ortho-aminobenzamide Hsp90 inhibitors is reported. A basic nitrogen within the tether linking the aniline nitrogen atom to a tetrahydroindolone moiety allowed access to compounds with good physical properties. Important structure-activity relationship information was obtained from this series which led to the discovery of a soluble and stable compound which is potent in an Hsp90 binding and cell-proliferation assay.

Discovery of a macrocyclic o-aminobenzamide Hsp90 inhibitor with heterocyclic tether that shows extended biomarker activity and in vivo efficacy in a mouse xenograft model.

A novel series of macrocyclic ortho-aminobenzamide Hsp90 inhibitors is reported. In continuation of our research, heterocycle-containing tethers were explored with the intent to further improve potency and minimize hERG liabilities. This effort culminated in the discovery of compound 10, which efficiently suppressed proliferation of HCT116 and U87 cells. This compound showed prolonged Hsp90-inhibitory activity at least 24h post-administration consistent with elevated and prolonged exposure in the tumor. When studied in a xenograft model, the compound demonstrated significant suppression of tumor growth.

Macrocyclic lactams as potent Hsp90 inhibitors with excellent tumor exposure and extended biomarker activity.

A novel series of macrocyclic ortho-aminobenzamide Hsp90 inhibitors is reported. In continuation of our research in this area, macrocyclic amides and lactams were explored to reduce the risk of hERG liabilities. This effort culminated in the discovery of compound 38, which showed a favorable in vitro profile, and efficiently suppressed proliferation of several relevant cell lines. This compound showed prolonged Hsp90-inhibitory activity at least 24 h post-administration, consistent with elevated and prolonged exposure in the tumor.

Optimization of 7-alkene-3-quinolinecarbonitriles as Src kinase inhibitors.

The 7-alkene-3-quinolinecarbonitrile 20, a potent inhibitor of Src enzymatic and cellular activity with IC(50) values of 2.1 and 58 nM, respectively, had comparable efficacy to bosutinib in a colon tumor xenograft study.

Inhibition of Src kinase activity by 4-anilino-5,10-dihydro-pyrimido[4,5-b]quinolines

4-(2,4-Dichloro-5-methoxy)anilino-5,10-dihydropyrimido[4,5-b]quinolines are potent inhibitors of Src kinase and Src cellular activity while having no effect on Fyn cellular activity. The corresponding 4-(2,4-dichloro-5-methoxy)anilino-pyrimido[4,5-b]quinolines are much less effective Src inhibitors.

A cell-based screen for inhibitors of protein folding and degradation

Cancer cells are exposed to external and internal stresses by virtue of their unrestrained growth, hostile microenvironment, and increased mutation rate. These stresses impose a burden on protein folding and degradation pathways and suggest a route for therapeutic intervention in cancer. Proteasome and Hsp90 inhibitors are in clinical trials and a 20S proteasome inhibitor, Velcade, is an approved drug. Other points of intervention in the folding and degradation pathway may therefore be of interest. We describe a simple screen for inhibitors of protein synthesis, folding, and proteasomal degradation pathways in this paper. The molecular chaperone-dependent client v-Src was fused to firefly luciferase and expressed in HCT-116 colorectal tumor cells. Both luciferase and protein tyrosine kinase activity were preserved in cells expressing this fusion construct. Exposing these cells to the Hsp90 inhibitor geldanamycin caused a rapid reduction of luciferase and kinase activities and depletion of detergent-soluble v-Src::luciferase fusion protein. Hsp70 knockdown reduced v-Src::luciferase activity and, when combined with geldanamycin, caused a buildup of v-Src::luciferase and ubiquitinated proteins in a detergent-insoluble fraction. Proteasome inhibitors also decreased luciferase activity and caused a buildup of phosphotyrosine-containing proteins in a detergent-insoluble fraction. Protein synthesis inhibitors also reduced luciferase activity, but had less of an effect on phosphotyrosine levels. In contrast, certain histone deacetylase inhibitors increased luciferase and phosphotyrosine activity. A mass screen led to the identification of Hsp90 inhibitors, ubiquitin pathway inhibitors, inhibitors of Hsp70/Hsp40-mediated refolding, and protein synthesis inhibitors. The largest group of compounds identified in the screen increased luciferase activity, and some of these increase v-Src levels and activity. When used in conjunction with appropriate secondary assays, this screen is a powerful cell-based tool for studying compounds that affect protein synthesis, folding, and degradation.

4-Anilino-7-pyridyl-3-quinolinecarbonitriles as Src kinase inhibitors.

A series of 4-anilino-7-pyridyl-3-quinolinecarbonitriles was prepared as Src kinase inhibitors. A systematic SAR study of substitutions on both the pyridine ring and the 3-quinolinecarbonitrile core established the requirements for optimal activity. The lead compound, 17, showed potent activity in both the Src enzyme assay and cell assays, and demonstrated in vivo anti-tumor activity in a xenograft model.